PROJECT SUMMARY More than 3 million US women are at risk of exposing their fetus to alcohol during pregnancy which may lead to cardiac defects since heart is the first organ to be established during development and is highly vulnerable to environmental insults such as alcohol exposure. Alcohol exposure is also associated with serious long-term complications of cardiac anomalies in adults. Studies of alcohol exposure have traditionally relied on animal models and primary cardiomyocytes (CMs) from animals which have provided valuable insights into the toxicity of alcohol. However, these models have well-known limitations due to physiological differences from human primary CMs which are difficult to obtain and have limited growth capacity. In addition, animal models are not amenable for high-throughput drug screening. Thus, a study on physiologically relevant human cells will be highly valuable to understand molecular mechanisms of alcohol-induced cardiac defects and to identify novel therapies. CMs derived from human pluripotent stem cells (hPSC-CMs) have many features similar to human primary CMs, can be produced in large quantities and maintained in long-term cultures, and can be adapted for high-throughput platforms. In our ongoing experiments, we have found that (1) microscale tissue engineering generates size- controlled 3D cardiac spheres of hPSC-CMs with high purity and improved maturation; (2) hPSC-CMs can be used for high-throughput screening of small molecules; (3) hPSC-CMs recapitulate disease phenotypes and clinically observed drug responses; and (4) exposure of hPSC-CMs to ethanol induces cytotoxicity, oxidative stress, and abnormal calcium handling. Given these findings, we hypothesize that hPSC-CMs, particularly those organized in 3D cardiac spheres, provide a novel and physiologically relevant system for the study of alcohol-induced cardiac defects and that novel molecular insights underlying alcohol-induced cardiac defects can be identified through a comprehensive study on the exposure of hPSC-CMs to ethanol. We will systematically characterize defects in hPSC-CMs following ethanol exposure and perform comprehensive transcriptomics, secretomics and metabolomics analyses to identify novel molecules/pathways underlying ethanol exposure. In addition, we will evaluate novel therapeutic approaches using hPSC-CMs. We expect that our proposed study using hPSC-CMs will help accelerate the development of targeted and effective therapies for alcohol-induced cardiac defects.